title: Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering.
abstract: BACKGROUND
  The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production.
  
  
  RESULTS
  An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate.
  
  
  CONCLUSIONS
  An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates.
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium.|Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium.
authors.forename: Mekonnen M
authors.initials: MM
authors.lastname: Demeke
authors.affiliation: Institute of Molecular Biosciences, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Dietz
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium.|Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Li
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium.|Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium.
authors.forename: María R
authors.initials: MR
authors.lastname: Foulquié-Moreno
authors.affiliation: Department of Chemical Engineering, Lund University, P.O. Box 124, 22100 Lund, Sweden.
authors.forename: Sarma
authors.initials: S
authors.lastname: Mutturi
authors.affiliation: Laboratory of Enzyme, Fermentation and Brewing Technology, KAHO Sint-Lieven University College, KU Leuven Association, Gebroeders De Smetstraat 1, 9000, Ghent, Flanders, Belgium.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Deprez
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium.|Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Den Abt
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium.|Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium.
authors.forename: Beatriz M
authors.initials: BM
authors.lastname: Bonini
authors.affiliation: Department of Chemical Engineering, Lund University, P.O. Box 124, 22100 Lund, Sweden.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Liden
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium.|Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium.
authors.forename: Françoise
authors.initials: F
authors.lastname: Dumortier
authors.affiliation: Laboratory of Enzyme, Fermentation and Brewing Technology, KAHO Sint-Lieven University College, KU Leuven Association, Gebroeders De Smetstraat 1, 9000, Ghent, Flanders, Belgium.
authors.forename: Alex
authors.initials: A
authors.lastname: Verplaetse
authors.affiliation: Institute of Molecular Biosciences, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Boles
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium.|Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium.
authors.forename: Johan M
authors.initials: JM
authors.lastname: Thevelein